Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels–I. Identification of calmodulin and the b subunit of calcineurin

At least three Ca²⁺-binding proteins were detected in rat cortex by ⁴⁵Ca²⁺ autoradiography of two-dimensional electrophoretograms. The identities of two of these Ca²⁺-binding proteins were determined to be calmodulin and the B subunit of calcineurin. The identification was based upon the following criteria: (1) co-localization on polyacrylamide gels with the appropriate purified proteins, (2) staining of nitrocellulose blots with specific antisera for calmodulin and calcineurin and (3) ability to bind Ca²⁺. This information is useful in that it identifies two major brain proteins visible on silver-stained two-dimensional polyacrylamide gels. In addition, this data reveals the location of an unidentified Ca²⁺-binding protein of molecular weight ∼ 18,000 Da and pI 5.4 on these gels.     

A method for measuring anion transfer across red cell membranes by continuous monitoring of fluorescence

A method was developed for the identification of Ca²⁺-binding proteins after electrophoresis on polyacrylamide gels. The method involves equilibration of the gel with ⁴⁵Ca either during or after electrophoresis, followed by visualization of the ⁴⁵Ca-binding proteins by autoradiography.     

Inhibition of the Ca2+-and phospholipid-dependent protein kinase by a novel Mr 17,000 Ca2+-binding protein

A novel M_r 17,000 Ca²⁺-binding protein isolated from bovine brain was found to be a potent inhibitor of the Ca²⁺- and phospholipid-dependent protein kinase (protein kinase C), also isolated from bovine brain. Halfmaximal inhibition by this calciprotein of the initial rate of phosphorylation of histone III-S by protein kinase C occurred at a calciprotein concentration of 2.2 μM under standard conditions. Comparison of the effects of a number of Ca²⁺-binding proteins on protein kinase C activity indicated that the M_r 17,000 Ca²⁺-binding protein was the most potent inhibitor, followed by the intestinal Ca²⁺-binding protein and calcineurin. Calmodulin, troponin C, S-100 protein and a M_r 21,000 Ca²⁺-binding protein of bovine brain were relatively weak inhibitors of protein kinase C. The inhibitory effect of the M_r 17,000 Ca²⁺-binding protein was apparently not due to its interaction with phospholipid or the basic protein substrate and therefore appears to be due to a direct effect on the protein kinase C. These observations suggest that the novel M_r 17,000 Ca²⁺-binding protein, and possibly other Ca²⁺-binding proteins, may play a physiological role in regulating the activity of protein kinase C.     

A rapid induction by elicitors of the mRNA encoding CCD-1, a 14 kDa Ca2+-binding protein in wheat cultured cells

Intracellular Ca²⁺ has been implicated in the signal transduction processes during the development of the plant defense system against fungal pathogens. From wheat cultured cells that had been treated with the elicitor derived from Typhula ishikariensis, the ccd-1 gene encoding a 14 kDa Ca²⁺-binding protein with an acidic amphiphilic feature was isolated. The ccd-1-encoded protein (CCD-1) shares homology to the C-terminal half domain of centrin, a Ca²⁺-binding protein conserved in eukaryotes. Unlike typical eukaryotic centrins, CCD-1 contains only one Ca²⁺-binding loop, which corresponds to the one in the fourth EF-hand from the N-terminus of centrin. The recombinant CCD protein expressed in Escherichia coli bound to a phenyl-Sepharose column in the presence of Ca²⁺ and was eluted out by EGTA. It also showed a Ca²⁺-dependent electrophoretic mobility shift on the non-denaturing polyacrylamide gel. The ccd-1 mRNA expression was rapidly induced by treatment with fungal and chitosan oligosaccharide elicitors, implying that it might have a role in transducing Ca²⁺ signals provoked by the elicitors. The expression of the ccd-1 mRNA was induced by treatment with A23187, and the induction was suppressed by La³⁺ or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA). This study suggests the involvement of intracellular Ca²⁺ in the elicitor-induced mRNA expression of a novel class of Ca²⁺-binding proteins conserved in higher plants.     

The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage

Effective control of the Ca²⁺ homeostasis in any living cell is paramount to coordinate some of the most essential physiological processes, including cell division, morphological differentiation, and intercellular communication. Therefore, effective homeostatic mechanisms have evolved to maintain the intracellular Ca²⁺ concentration at physiologically adequate levels, as well as to regulate the spatial and temporal dynamics of Ca²⁺signaling at subcellular resolution. Members of the superfamily of EF-hand Ca²⁺-binding proteins are effective to either attenuate intracellular Ca²⁺ transients as stochiometric buffers or function as Ca²⁺ sensors whose conformational change upon Ca²⁺ binding triggers protein-protein interactions, leading to cell state-specific intracellular signaling events. In the central nervous system, some EF-hand Ca²⁺-binding proteins are restricted to specific subtypes of neurons or glia, with their expression under developmental and/or metabolic control. Therefore, Ca²⁺-binding proteins are widely used as molecular markers of cell identity whilst also predicting excitability and neurotransmitter release profiles in response to electrical stimuli. Secretagogin is a novel member of the group of EF-hand Ca²⁺-binding proteins whose expression precedes that of many other Ca²⁺-binding proteins in postmitotic, migratory neurons in the embryonic nervous system. Secretagogin expression persists during neurogenesis in the adult brain, yet becomes confined to regionalized subsets of differentiated neurons in the adult central and peripheral nervous and neuroendocrine systems. Secretagogin may be implicated in the control of neuronal turnover and differentiation, particularly since it is re-expressed in neoplastic brain and endocrine tumors and modulates cell proliferation in vitro. Alternatively, and since secretagogin can bind to SNARE proteins, it might function as a Ca²⁺ sensor/coincidence detector modulating vesicular exocytosis of neurotransmitters, neuropeptides or hormones. Thus, secretagogin emerges as a functionally multifaceted Ca²⁺-binding protein whose molecular characterization can unravel a new and fundamental dimension of Ca²⁺signaling under physiological and disease conditions in the nervous system and beyond.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Effects of Metal-Binding Properties of Human Kv Channel-Interacting Proteins on their Molecular Structure and Binding with Kv4.2 Channel

The goal of the present study is to explore whether Ca²⁺ and Mg²⁺-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca²⁺-binding sites, respectively, and only one type of Mg²⁺-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca²⁺, but the binding affinity for Mg²⁺ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg²⁺ and Ca²⁺. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg²⁺ and Ca²⁺. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg²⁺- and Ca²⁺-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.     

Prediction and Analysis of Canonical EF Hand Loop and Qualitative Estimation of Ca2+ Binding Affinity

The diversity of functions carried out by EF hand-containing calcium-binding proteins is due to various interactions made by these proteins as well as the range of affinity levels for Ca²⁺ displayed by them. However, accurate methods are not available for prediction of binding affinities. Here, amino acid patterns of canonical EF hand sequences obtained from available crystal structures were used to develop a classifier that distinguishes Ca²⁺-binding loops and non Ca²⁺-binding regions with 100% accuracy. To investigate further, we performed a proteome-wide prediction for E. histolytica, and classified known EF-hand proteins. We compared our results with published methods on the E. histolytica proteome scan, and demonstrated our method to be more specific and accurate for predicting potential canonical Ca²⁺-binding loops. Furthermore, we annotated canonical EF-hand motifs and classified them based on their Ca²⁺-binding affinities using support vector machines. Using a novel method generated from position-specific scoring metrics and then tested against three different experimentally derived EF-hand-motif datasets, predictions of Ca²⁺-binding affinities were between 87 and 90% accurate. Our results show that the tool described here is capable of predicting Ca²⁺-binding affinity constants of EF-hand proteins. The web server is freely available at http://202.41.10.46/calb/index.html.     

Insights into modulation of calcium signaling by magnesium in calmodulin, troponin C and related EF-hand proteins

The Ca²⁺-binding helix-loop-helix structural motif called “EF-hand” is a common building block of a large family of proteins that function as intracellular Ca²⁺-receptors. These proteins respond specifically to micromolar concentrations of Ca²⁺ in the presence of ~1000-fold excess of the chemically similar divalent cation Mg²⁺. The intracellular free Mg²⁺ concentration is tightly controlled in a narrow range of 0.5-1.0 mM, which at the resting Ca²⁺ levels is sufficient to fully or partially saturate the Ca²⁺-binding sites of many EF-hand proteins. Thus, to convey Ca²⁺ signals, EF-hand proteins must respond differently to Ca²⁺ than to Mg²⁺. In this review the structural aspects of Mg²⁺ binding to EF-hand proteins are considered and interpreted in light of the recently proposed two-step Ca²⁺-binding mechanism (Grabarek, Z., J. Mol. Biol., 2005, 346, 1351). It is proposed that, due to stereochemical constraints imposed by the two-EF-hand domain structure, the smaller Mg²⁺ ion cannot engage the ligands of an EF-hand in the same way as Ca²⁺ and defaults to stabilizing the apo-like conformation of the EF-hand. It is proposed that Mg²⁺ plays an active role in the Ca²⁺-dependent regulation of cellular processes by stabilizing the “off state” of some EF-hand proteins, thereby facilitating switching off their respective target enzymes at the resting Ca²⁺ levels. Therefore, some pathological conditions attributed to Mg²⁺ deficiency might be related to excessive activation of underlying Ca²⁺-regulated cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Ca2+ -binding proteins and contractility of the infraciliary lattice in Paramecium

The infraciliary lattice (ICL) is the innermost cortical cytoskeletal network of Paramecium. Its meshes which run around the proximal end of basal bodies form a continuous contractile network beneath the cell surface. We had previously shown that the network, which could be recovered in a contracted form and selectively solubilized by EGTA from an ICL-enriched cell fraction, was principally composed of 23–24 kDa polypeptides cross-reacting with antibodies raised against the 22 kDa Ca²⁺ -binding proteins of the ecto-endoplasmic boundary (EEB), a contractile cytoskeletal network of another ciliate Isotricha prostoma. We show here 1) that the ICL also comprises a 220 kDa polypeptide; 2) that the 23–24 kDa polypeptides are resolved in 2D gels into 11 spots of acidic pI, 7 of which are both Ca²⁺ -binding and cross-reacting with the anti EEB polypeptides; 3) that the network displays a high Ca²⁺ -affinity as the treshold for solubilization/co-precipitation of both high and low MW polypeptides is around 10⁻⁸ M free Ca²⁺ ; 4) that in vivo contraction of the network occurs upon physiological increase of internal calcium concentration. The likely phylogenetic relationships of the 23–24 kDa ICL polypeptides with the calmodulin related family of Ca²⁺ -modulated polypeptides and the functions of the ICL in cell contractility and Ca²⁺ homeostasis are discussed.     

Spine neck geometry determines spino-dendritic cross-talk in the presence of mobile endogenous calcium binding proteins

Dendritic spines are thought to compartmentalize second messengers like Ca²⁺. The notion of isolated spine signaling, however, was challenged by the recent finding that under certain conditions mobile endogenous Ca²⁺-binding proteins may break the spine limit and lead to activation of Ca²⁺-dependent dendritic signaling cascades. Since the size of spines is variable, the spine neck may be an important regulator of this spino-dendritic crosstalk. We tested this hypothesis by using an experimentally defined, kinetic computer model in which spines of Purkinje neurons were coupled to their parent dendrite by necks of variable geometry. We show that Ca²⁺ signaling and calmodulin activation in spines with long necks is essentially isolated from the dendrite, while stubby spines show a strong coupling with their dendrite, mediated particularly by calbindin D28k. We conclude that the spine neck geometry, in close interplay with mobile Ca²⁺-binding proteins, regulates the spino-dendritic crosstalk.     

Analysis of EF-hand-containing proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

In plants, calcium (Ca²⁺) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca²⁺]_cyt), which in turn is thought to regulate cellular and developmental processes via Ca²⁺-binding proteins. Three out of the four classes of Ca²⁺-binding proteins in plants contain Ca²⁺-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca²⁺ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.     

Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells

In this paper we review some of the large quantities of information currently available concerning the identification, structure and function of Ca²⁺-binding proteins of endoplasmic and sarcoplasmic reticulum membranes. The review places particular emphasis on identification and discussion of Ca²⁺ storage proteins in these membranes. We believe that the evidence reviewed here supports the contention that the Ca²⁺-binding capacity of both calsequestrin and calreticulin favor their contribution as the major Ca²⁺-binding proteins of muscle and nonmuscle cells, respectively. Other Ca²⁺-binding proteins discovered in both endoplasmic reticulum and sarcoplasmic reticulum membranes probably contribute to the overall Ca²⁺ storage capacity of these membrane organelles, and they also play other important functional role such as posttranslational modification of newly synthesized proteins, a cytoskeletal (structural) function, or movement of Ca²⁺ within the lumen of the sarcoplasmic/endoplasmic reticulum towards the storage sites.Abbreviations SR Sarcoplasmic Reticulum - ER Endoplasmic Reticulum - InsP₃ Inositol 1,4,5-trisphosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - PDI Protein Disulphide Isomerase - T₃BP Thyroid Hormone Binding Protein - Grp Glucose regulated proteins - HCP Histidine-rich Ca²⁺ binding Protein - LDL Low Density Lipoprotein     

Vitamin D-3 induces the de novo synthesis of calmodulin in Phaseolus vulgaris root segments growing in vitro

Vitamin D-3 affects growth and Ca²⁺ transport in Phaseolus vulgaris roots by a mechanism dependent on de novo protein synthesis. The objective of this work was to identify the protein(s) induced by the sterol. Phaseolus vulgaris root segments cultured in vitro were used. Protein extracts of control cultures and cultures treated with 10⁻⁹ M vitamin D-3 for 2 h labelled with [¹⁴C]leucine and [³H]leucine, respectively, were mixed and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Examination of ³H:¹⁴C ratios in slices of gels revealed that vitamin D-3 stimulates production of a protein which exhibited Ca²⁺-dependent electrophoretic mobility. The apparent molecular weight was 14500 in the presence of 1 mM Ca²⁺ and 18 000 in the presence of 5 mM EGTA. This protein was heat- and acid-stable, bound ⁴⁵Ca²⁺ and had an isoelectric pH of 3.8-4.1. These data are consistent with the properties of calmodulin. In agreement with these observations, higher levels of calmodulin were detected in roots treated with vitamin D-3 than in control roots by means of assays based on the activation of calmodulin-dependent enzymes. Moreover, estimation of calmodulin relative synthesis and degradation rates in roots cultures in the absence and presence of vitamin D-3 indicated that the sterol increases calmodulin synthesis without affecting its degradation. In addition, the results suggest that the synthesis of other low-molecular-weight Ca²⁺-binding proteins may be affected by vitamin D-3.     

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site

In order to determine whether polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca²⁺ + Mg²⁺-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca²⁺ + Mg²⁺-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca²⁺ + Mg²⁺-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca²⁺-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca²⁺. Its lack of cross-reactivity with proteins that do not bind Ca²⁺ suggests that it has specificity for antigenic determinants that make up the Ca²⁺-binding sites in several Ca²⁺-binding proteins including the Ca²⁺ + Mg²⁺-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.     

Ca2+-Binding S100 Proteins in the Central Nervous System

A number of neurodegenerative disorders have been attributed to abberrations of intracellular Ca²⁺ homeostasis regulated by Ca²⁺-binding proteins. This chapter will focus on the S100B and S100A6 proteins, which are highly expressed in the central nervous system. Their protein structures, localizations, and association with brain pathology as well as their potential use in clinical diagnostics will be discussed.     

NMR structure and calcium-binding properties of the tellurite resistance protein TerD from Klebsiella pneumoniae

The tellurium oxyanion TeO₃²⁻ has been used in the treatment of infectious diseases caused by mycobacteria. However, many pathogenic bacteria show tellurite resistance. Several tellurite resistance genes have been identified, and these genes mediate responses to diverse extracellular stimuli, but the mechanisms underlying their functions are unknown. To shed light on the function of KP-TerD, a 20.5 -kDa tellurite resistance protein from a plasmid of Klebsiella pneumoniae, we have determined its three-dimensional structure in solution using NMR spectroscopy. KP-TerD contains a β-sandwich formed by two five-stranded β-sheets and six short helices. The structure exhibits two negative clusters in loop regions on the top of the sandwich, suggesting that KP-TerD may bind metal ions. Indeed, thermal denaturation experiments monitored by circular dichroism and NMR studies reveal that KP-TerD binds Ca²⁺. Inductively coupled plasma-optical emission spectroscopy shows that the binding ratio of KP-TerD to Ca²⁺ is 1:2. EDTA (ethylenediaminetetraacetic acid) titrations of Ca²⁺-saturated KP-TerD monitored by one-dimensional NMR yield estimated dissociation constants of 18  and 200 nM for the two Ca²⁺-binding sites of KP-TerD. NMR structures incorporating two Ca²⁺ ions define a novel bipartite Ca²⁺-binding motif that is predicted to be highly conserved in TerD proteins. Moreover, these Ca²⁺-binding sites are also predicted to be present in two additional tellurite resistance proteins, TerE and TerZ. These results suggest that some form of Ca²⁺ signaling plays a crucial role in tellurite resistance and in other responses of bacteria to multiple external stimuli that depend on the Ter genes.     

Calcium-binding of synaptosomes isolated from rat brain cortex

Summary Ruthenium red combines with isolated synaptosomes, resulting in strong inhibition of their Ca²⁺-binding. In isotonic saline media, however, the dye-induced inhibition of Ca²⁺-binding is significantly greater than that expected for the amount of bound dye and Hill's exponent of the Ca²⁺-binding decreases to 1 with an increase in the amount of the dye bound. On the other hand in isotonic mannitol-sucrose solution, inhibition of synaptosomal Ca²⁺-binding brought about by the dye is proportional to the amount of dye bound. Based on these results, the effects of the dye on the co-operative nature of synaptosomal Ca²⁺-binding is discussed.     

STIM proteins,Orai1, and gene expression

Cytoplasmic Ca²⁺ is an universal intracellular messenger that activates cellular responses over a broad temporal range, from neurotransmitter release to cell growth and proliferation.^1,2 Inherent to the use of the multifarious Ca²⁺ signal is the question of specificity: how can some Ca²⁺-dependent responses be activated in a cell and not others? A rise in cytoplasmic Ca²⁺ can evoke a response either by binding directly to the target (as occurs with certain Ca²⁺-activated K⁺ and Cl⁻ channels, for example) or through recruitment of intermediary proteins, such as calmodulin and troponin C. A substantial body of evidence has now established that Ca²⁺-binding proteins differ both in their affinities for Ca²⁺ and in their on- and off-rates for Ca²⁺ binding/unbinding. Furthermore, different Ca²⁺-binding proteins often occupy distinct locations within the cell. Therefore, the size, kinetics and spatial profile of a cytoplasmic Ca²⁺ signal are all important in determining which Ca²⁺-dependent response will be activated, when and for how long.³     

The Ca2+-binding protein calretinin is selectively enriched in a subpopulation of the epithelial rests of Malassez

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca²⁺-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca²⁺-binding proteins in the ERM has not so far been characterized. Among the three Ca²⁺-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca²⁺ is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca²⁺ is regulated only by calretinin. The expression of Ca²⁺-binding proteins is restricted in a developmental manner in the ERM.